Genetic variation and demographic history of the Haplochromis laparogramma group of Lake Victoria-An analysis based on SINEs and mitochondrial DNA

More than 500 endemic haplochromine cichlid species inhabit Lake Victoria. This striking species diversity is a classical example of recent explosive adaptive radiation thought to have happened within the last similar to 15,000 years. In this study, we examined the population structure and historical demography of 3 pelagic haplochromine cichlid species that resemble in morphology and have similar niche, Haplochromis (Yssichromis) laparogramma, Haplochromis (Y.) pyrrhocephalus, and Haplochromis (Y.) sp. "glaucocephalus". We investigated the sequences of the mitochondrial DNA control region and the insertion patterns of short interspersed elements (SINEs) of 759 individuals. We show that sympatric forms are genetically differentiated in 4 of 6 cases, but we also found apparent weakening of the genetic differentiation in areas with turbid water. We estimated the timings of population expansion and species divergence to coincide with the refilling of the lake at the Pleistocene/Holocene boundary. We also found that estimates can be altered significantly by the choice of the shape of the molecular clock. If we employ the nonlinear clock model of evolutionary rates in which the rates are higher towards the recent, the population expansion was dated at around the event of desiccation of the lake ca. 17,000 YBP. Thus, we succeeded in clarifying the species and population structure of closely related Lake Victoria cichlids and in showing the importance of applying appropriate clock calibrations in elucidating recent evolutionary events. (C) 2009 Elsevier B.V. All rights reserved.

The 'triple contrast' method in experimental wound ballistics and backspatter analysis.

In practical forensic casework, backspatter recovered from shooters' hands can be an indicator of self-inflicted gunshot wounds to the head. In such cases, backspatter retrieved from inside the barrel indicates that the weapon found at the death scene was involved in causing the injury to the head. However, systematic research on the aspects conditioning presence, amount and specific patterns of backspatter is lacking so far. Herein, a new concept of backspatter investigation is presented, comprising staining technique, weapon and target medium: the 'triple contrast method' was developed, tested and is introduced for experimental backspatter analysis. First, mixtures of various proportions of acrylic paint for optical detection, barium sulphate for radiocontrast imaging in computed tomography and fresh human blood for PCR-based DNA profiling were generated (triple mixture) and tested for DNA quantification and short tandem repeat (STR) typing success. All tested mixtures yielded sufficient DNA that produced full STR profiles suitable for forensic identification. Then, for backspatter analysis, sealed foil bags containing the triple mixture were attached to plastic bottles filled with 10 % ballistic gelatine and covered by a 2-3-mm layer of silicone. To simulate backspatter, close contact shots were fired at these models. Endoscopy of the barrel inside revealed coloured backspatter containing typable DNA and radiographic imaging showed a contrasted bullet path in the gelatine. Cross sections of the gelatine core exhibited cracks and fissures stained by the acrylic paint facilitating wound ballistic analysis.

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction.

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19-parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self-devised pentaplex PCR generating an "integrity score" (Si ). STR typing success rate was then assessed for selected cases. DNA yield and Si differed significantly between tissues with kidney on average exhibiting the highest Si values. Statistical analysis revealed that nine parameters were significantly and positively correlated with Si . The observed values for each of these nine parameters were summed up to generate a putrefaction score (Sp ) for each sample. Our results show that STR typing success rate can be predicted based on Sp before expensive multiplex STR profiling is performed.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.